U.S. patent application number 17/042122 was filed with the patent office on 2022-07-21 for snp marker combination and identification method for pudong white pigs and raw meat products.
The applicant listed for this patent is ZHEJIANG UNIVERSITY. Invention is credited to Yuchun PAN, Qishan WANG, Zhen WANG, Zhe ZHANG.
Application Number | 20220228225 17/042122 |
Document ID | / |
Family ID | 1000006290674 |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220228225 |
Kind Code |
A1 |
PAN; Yuchun ; et
al. |
July 21, 2022 |
SNP MARKER COMBINATION AND IDENTIFICATION METHOD FOR PUDONG WHITE
PIGS AND RAW MEAT PRODUCTS
Abstract
The present invention discloses a SNP marker combination and an
identification method for Pudong white pigs and raw meat products
thereof, including: extracting genomic DNAs of raw pork or meat
products, performing agarose gel electrophoresis and Sanger
sequencing after PCR amplification, and identifying Pudong white
pigs and meat products thereof according to SNP genotypes of
characteristic loci of sequencing results; as for the Sanger
sequencing, its identification loci are as follows: specific
mutations occur at loci of pig18-52722267, pig8-146130825,
pig9-10041850 and pig13-213464983. The present invention solves the
problem that there is no identification method related to Pudong
white pigs and meat products thereof in the prior art.
Inventors: |
PAN; Yuchun; (Hangzhou City,
Zhejiang Province, CN) ; WANG; Qishan; (Hangzhou
City, Zhejiang Province, CN) ; ZHANG; Zhe; (Hangzhou
City, Zhejiang Province, CN) ; WANG; Zhen; (Hangzhou
City, Zhejiang Province, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZHEJIANG UNIVERSITY |
Hangzhou City, Zhejiang Province |
|
CN |
|
|
Family ID: |
1000006290674 |
Appl. No.: |
17/042122 |
Filed: |
December 31, 2019 |
PCT Filed: |
December 31, 2019 |
PCT NO: |
PCT/CN2019/130973 |
371 Date: |
September 26, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6888 20130101 |
International
Class: |
C12Q 1/6888 20060101
C12Q001/6888 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2019 |
CN |
201910463234.2 |
Claims
1. A SNP marker combination for Pudong white pigs and raw meat
products, comprising pig18-52722267, pig8-146130825, pig9-10041850
and pig13-213464983, wherein the pig18-52722267 represents a
52722266.sup.th locus of pig chromosome No. 18, the pig8-146130825
represents a 146130825.sup.th locus of pig chromosome No. 8, the
pig9-10041850 represents a 10041850.sup.th locus of pig chromosome
No. 9, and the pig13-213464983 represents a 213464983.sup.th locus
of pig chromosome No. 13.
2. A method for identifying Pudong white pigs and raw meat products
thereof based on the SNP marker combination according to claim 1,
comprising: firstly extracting genomic DNAs of a raw pork or a meat
product to be identified, then performing agarose gel
electrophoresis and Sanger sequencing after PCR amplification to
obtain information of each detection locus of the SNP marker
combination, wherein a Pudong white pig and a meat product thereof
are identified when specific mutations occur at any three loci of
the SNP marker combination.
3. The method according to claim 2, wherein as for the PCR
amplification, sequences of upstream and downstream primers of the
pig18-52722267 are shown in SEQ ID No.1 and SEQ ID No.2, sequences
of upstream and downstream primers of the pig8-146130825 are shown
in SEQ ID No.3 and SEQ ID No.4, sequences of upstream and
downstream primers of the pig9-10041850 are shown in SEQ ID No.5
and SEQ ID No.6, and sequences of upstream and downstream primers
of the pig13-2134664983 are shown in SEQ ID No. 7 and SEQ ID
No.8.
4. The method according to claim 2, wherein comparison information
of identification loci of sequencing products is shown in the
following table: TABLE-US-00006 SNP REF ALT pig18-52722267 T C
pig8-146130825 G T pig9-10041850 G A pig13-213464983 C T
wherein REF represents a reference genotype and ALT represents a
mutation genotype.
5. The method according to claim 4, wherein the information of
identification loci is: mutation genotypes and reference genotypes
of detection loci; when a reference genotype appears in a detection
locus of a pig to be detected, the locus is determined to have no
identification significance; and when a mutation genotype appears
in a detection locus of the pig to be detected, the locus is
determined to have identification significance.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of food safety
monitoring, and in particular to a SNP marker combination and an
identification method for Pudong white pigs and raw meat
products.
BACKGROUND
[0002] Pudong white pigs are mainly distributed in Nanhui and
Chuansha of Shanghai. They are characterized by a high reproduction
rate and early sexual maturity, with an average of about 12 litters
per birth. After castration, they are quiet and motionless, and
suitable for soft-ring breeding. Before 1950s, they were the
dominant breed in Chuansha County. Because of their delicious meat,
they are widely welcomed by consumers. Pudong white pigs are
similar in shape and appearance to Western Landrace and Yorkshire
pigs, and there are cases in which Western pig breeds pretend to be
Pudong white pigs in production. On the other hand, the
distribution area of Pudong white pigs overlaps with that of Taihu
Pigs (Erhualian Pigs, Meishan Pigs, Fengjing Pigs Shawutou Pigs, Mi
Pigs and Jiaxing Black Pigs), which leads to confusion in
production. It is very difficult to distinguish Pudong white pigs
from other breeds of pigs by traditional methods, especially the
slaughtered divided pigs and their processed meat products. With
the development of sequencing technology, molecular markers have
developed from the first generation of restriction fragment length
polymorphism (RFLP) and the second generation of variable number
Simple Sequence Repeat (SSR) to single nucleotide polymorphism
(SNP). Compared with the previous two generations, the
third-generation molecular marker SNP has the advantages of
abundant variation, low requirement for DNA samples, high
stability, accurate determination, simple detection method and high
throughput. At present, the third-generation molecular marker SNP
has been widely used in paternity testing, identification of animal
and plant varieties (strains), genetic breeding and other
fields.
SUMMARY
[0003] The purpose of the present invention is to provide a SNP
marker combination and an identification method for identifying
Pudong white pigs and raw meat products thereof, aiming at the
defects of the prior art. The third generation molecular marker
identification and Sanger sequencing technology are used to
identify Pudong white pigs and raw meat products thereof, which
solves the problem that there is no identification method related
to Pudong white pigs and their meat products in the prior art, and
provides a method and related special primers for identifying
Pudong white pigs and meat products thereof with accurate results,
simple operations and a low price.
[0004] The purpose of the present invention is realized by the
following technical solution:
[0005] A SNP marker combination for Pudong white pigs and raw meat
products comprises pig18-52722267, pig8-146130825, pig9-10041850
and pig13-213464983, wherein the pig18-52722267 represents a
52722266.sup.th locus of pig chromosome No. 18, the pig8-146130825
represents a 146130825.sup.th locus of pig chromosome No. 8, the
pig9-10041850 represents a 10041850.sup.th locus of pig chromosome
No. 9, and the pig13-213464983 represents a 213464983.sup.th locus
of pig chromosome No. 13.
[0006] Furthermore, a method for identifying Pudong white pigs and
raw meat products thereof based on the SNP marker combination
comprises : firstly extracting genomic DNAs of a raw pork or meat
product to be identified, then performing agarose gel
electrophoresis and Sanger sequencing after PCR amplification to
obtain information of each detection locus of the SNP marker
combination, wherein a Pudong white pig and a meat product thereof
are identified when specific mutations occur at any three loci of
the SNP marker combination.
[0007] Furthermore, as for the PCR amplification, sequences of
upstream and downstream primers of the pig18-52722267 are shown in
SEQ ID No.1 and SEQ ID No.2, sequences of upstream and downstream
primers of the pig8-146130825 are shown in SEQ ID No.3 and SEQ ID
No.4, sequences of upstream and downstream primers of the
pig9-10041850 are shown in SEQ ID No.5 and SEQ ID No.6, and
sequences of upstream and downstream primers of the
pig13-2134664983 are shown in SEQ ID No. 7 and SEQ ID No.8.
[0008] Furthermore, comparison information of identification loci
of sequencing products is shown in the following table:
TABLE-US-00001 SNP REF ALT pig18-52722267 T C pig8-146130825 G T
pig9-10041850 G A pig13-213464983 C T
[0009] in which, REF represents a reference genotype and ALT
represents a mutation genotype.
[0010] Furthermore, the information of identification loci is:
mutation genotypes and reference genotypes of detection loci; when
a reference genotype appears in a detection locus of a pig to be
detected, the locus is determined to have no identification
significance; and when a mutation genotype appears in a detection
locus of the pig to be detected, the locus is determined to have
identification significance.
[0011] The beneficial effect of the present invention is: compared
with the prior art, the present invention takes the unique SNP loci
of Pudong white pigs as the identification basis, studies the
identification method of Pudong white pigs from the molecular
level, and takes Sanger sequencing as the main molecular
identification method, which can distinguish Pudong white pigs from
common western pig breeds and local pig breeds in Taihu Basin, for
example, small Meishan pigs, Fengjing pigs and Middle Meishan pigs,
Erhualian pigs, Jiaxing black pigs, Mi pigs, Shawutou pigs,
Landraces, Yorkshire pigs, Durocs, Pietrains, Barkshire, etc.
DESCRIPTION OF EMBODIMENTS
[0012] The specific embodiments of the present invention will be
described in further detail below.
[0013] The present invention relates to a SNP marker combination of
Pudong white pigs and raw meat products, comprising pig18-52722267,
pig8-146130825, pig9-10041850 and pig13-213464983, wherein the
pig18-52722267 represents the 52722266.sup.th locus of pig
chromosome No. 18, the pig8-146130825 represents the
146130825.sup.th locus of pig chromosome No. 8, the pig9-10041850
represents the 10041850.sup.th locus of pig chromosome No. 9, and
the pig13-213464983 represents the 213464983.sup.th locus of pig
chromosome No. 13.
[0014] The present invention provides a method for identifying
Pudong white pigs and raw meat products thereof based on the above
SNP marker combination, comprising the following steps of: firstly
extracting genomic DNA of raw pork or meat products to be
identified, then performing agarose gel electrophoresis and Sanger
sequencing after PCR amplification to obtain information of each
detection locus of the SNP marker combination, wherein a Pudong
white pig and a meat product thereof are identified when specific
mutations occur at any three loci of the SNP marker
combination.
[0015] The primers involved in the PCR amplification are shown in
Table 1:
TABLE-US-00002 TABLE 1 Information about Primers and Products of
Amplification Loci SNP F R length F'-position pig18-52722267 SEQ ID
NO. 1: SEQ ID NO. 2: 314 160 GCGTTTTGGGGACTCGTGATA
ACTCTGCCGTTTCTCCTCCTA pig8-146130825 SEQ ID NO. 3: SEQ ID NO. 4:
AGAGGAGCGGGGTCTTTGC CGTTGTCAACTTTTGTCTACCT 525 119 CA pig9-10041850
SEQ ID NO. 5: SEQ ID NO. 6: 627 284 TAGATGTGAGCCCCAGCAGTT
ACTTCTGCTCCCCGCACCG pig13-213464983 SEQ ID NO. 7: SEQ ID NO. 8: 177
71 CACTCAGGATATTCACAATCTGG TTAAAACGACCACGGCAACTC
[0016] In the table, F represents an upstream primer, R represents
a downstream primer, length represents the length of a standard
product, and F'-position represents the position of a SNP locus in
an amplification product.
[0017] In the PCR amplification, the reaction system is template
DNA of 1 ng/uL, a primer of 1 uL, H.sub.2O of 3.8 uL and 2.times.
Taq PCR masrer mix of 50 ul; and/or the reaction procedure of the
PCR reaction is: pre-denaturation at 95C.degree. for 2 minutes,
pre-denaturation at 95C..degree. for 30 seconds, annealing at
60C..degree. for 30 seconds, extension at 72C..degree. for 1
minute, with a cycle number of 30 times, and extension at
72C..degree. for another 10 min minutes.
[0018] The mass concentration of the agarose gel is 2%.
[0019] The strip length of the gel electrophoresis is required as
shown in Table 1.
[0020] The comparison information of identification loci of
sequencing products is shown in Table 2:
TABLE-US-00003 TABLE 2 Comparison Information of Identification
Loci of Sequencing Products SNP REF ALT pig18-52722267 T C
pig8-146130825 G T pig9-10041850 G A pig13-213464983 C T
[0021] in which, REF represents a reference genotype and ALT
represents a mutation genotype.
[0022] Table 2 shows the mutation genotype and reference genotype
of each detection locus. A detection locus is considered to have no
identification significance when a reference genotype appears in
the locus of the pig to be detected, and the detection locus is
considered to have identification significance when a mutation
genotype appears in the locus of the pig to be detected. For
example, for a pig18-52722267 locus of pig A to be detected,
[0023] if the sequencing data is T, it is considered that pig A has
no identification significance at pig 18-5272267 locus; if the
sequencing data is C, it is considered that pig a has
identification significance at pig18-52722267 locus.
[0024] Since there is false positive misjudgment when a single
locus is used as the identification basis, and the misjudgment
probability is high, the present invention uses the locus
combination as the identification Marker.
[0025] Marker information of each breed identification locus
combination is shown in Table 3.
TABLE-US-00004 TABLE 3 Information about Identification Marker
Combination Breed Marker SNP combination Pudong Marker 1
pig18-52722267, pig8-146130825, white pig pig9-10041850 Marker 2
pig18-52722267, pig8-146130825, pig13-213464983 Marker 3
pig18-52722267, pig9-10041850, pig13-213464983 Marker 4
pig8-146130825, pig9-10041850, pig13-213464983
[0026] As shown in Table 3, Markers 1-4 are identification marker
combinations for Pudong white pigs. For example, when the pig A to
be detected has any one of the four marker combinations, it is
considered that the pig A to be detected is a Pudong white pig.
[0027] The Pudong white pork products refer to Pudong white pig
split meat and pickled products and cooked food products prepared
from Pudong white pigs as raw materials.
[0028] Five ear tissue samples of pigs to be detected were randomly
selected, and tissue DNA was extracted by a SDS method.
[0029] PCR amplification of DNA samples was carried out by
primers.
[0030] The reaction system was template DNA of 1 ng/uL, a primer of
1 uL, H.sub.2O of 3.8 uL and 2.times. Taq PCR masrer mix of 50 ul;
and/or the reaction procedure of the PCR reaction was:
pre-denaturation at 95C..degree. for 2 minutes, pre-denaturation at
95C..degree. for 30 seconds, annealing at 60.degree. C. for 30
seconds, extension at 72C..degree. for 1 minute, with a cycle
number of 30 times, and extension at 72C..degree. for another 10
min minutes.
[0031] The amplification results were detected by electrophoresis
with 2% of an agarose gel and 1.times. TAE of a buffer solution as
mediums. The gel electrophoresis conditions were: a current of 10
A, a voltage of 100v and time of 40 min. The amplification products
of different primer pairs were compared with the standard product
length in Table 1. If the product length is within the error range
and consistent with the standard product length, the amplification
result is considered qualified.
TABLE-US-00005 TABLE 4 SNP Polymorphism Analysis Table of Sample
Sequencing Results Sample/SNP REF ALT 1 2 3 4 5 pig18-52722267 T C
pig8-146130825 G T pig9-10041850 G A pig13-213464983 C T
[0032] In the table, REF represents a reference genotype, ALT
represents a mutation genotype, and represents a detected mutation
genotype.
[0033] As shown in Table 4, it can be seen from the sequencing data
that if only a single locus is used as the identification basis, a
pig to be detected belongs to multiple breeds at the same time.
[0034] Identification was carried out by using a Marker
combination: mutations were detected at pig18-52722267 in a pig No.
1, which did not conform to Marker information, and the pig No. 1
was identified as not a Pudong white pig; mutations were detected
at pig18-52722267, pig8-146130825, pig9-10041850 in a pig No. 2,
which conformed to the Marker information, and the pig No. 2 was
identified as a Pudong white pig; mutations were detected at
pig8-146130825 and pig9-10041850 in a pig No. 3, which did not
conform to the Marker information, and the pig No. 3 was identified
as not a Pudong white pig; mutations were detected at
pig18-52722267 and pig13-213464983 in a pig No. 4, which did not
conform to the Marker information, and the pig No. 4 was identified
as not a Pudong white pig; mutations were detected at
pig8-146130825, pig9-10041850 and pig13-213464983 in pig No. 5,
which conformed to Marker4, and the pig No. 5 was identified as a
Pudong white pig.
[0035] At present, there are no patents related to the
identification of Pudong white pigs and their meat products at home
and abroad. The invention of the third-generation molecular markers
to the identification of Pudong white pigs and their meat products
has filled the blank in the market and effectively solved the
problem of identification of Pudong white pigs. Compared with the
existing patents that use the first-generation molecular markers
(RFLP) and the second-generation molecular markers (SSR) to
identify pig breeds, this identification method has the advantages
of simpler operation, more accurate results, rapidness and high
efficiency. Meanwhile, the third-generation molecular marker SNP is
utilized in the present invention to overcome the defect of fewer
available loci for the molecular markers of the previous two
generations, and compared with the identification technology of the
previous two generations of molecular markers, the identification
method is also simplified.
[0036] The above specific implementation can be partially adjusted
by those skilled in the art in different ways without departing
from the principles and purposes of the present invention. The
protection scope of the present invention is subject to the claims
and not limited by the above specific implementation, and all
embodiments within its scope are subject to the present invention.
Sequence CWU 1
1
8121DNAArtificial Sequenceprimer 1gcgttttggg gactcgtgat a
21221DNAArtificial Sequenceprimer 2actctgccgt ttctcctcct a
21319DNAArtificial Sequenceprimer 3agaggagcgg ggtctttgc
19424DNAArtificial Sequenceprimer 4cgttgtcaac ttttgtctac ctca
24521DNAArtificial Sequenceprimer 5tagatgtgag ccccagcagt t
21619DNAArtificial Sequenceprimer 6acttctgctc cccgcaccg
19723DNAArtificial Sequenceprimer 7cactcaggat attcacaatc tgg
23821DNAArtificial Sequenceprimer 8ttaaaacgac cacggcaact c 21
* * * * *